We are studying the oxygen exchange properties of the actomyosin ATPase from rabbit muscle. When (18 O)-ATP is hydrolyzed by myosin in the absence of actin, total exchange (or washout) of the 18 O into water takes place so that the product phosphate has the same oxygen enrichment as the medium. When actin is added, the character of the reaction changes. A second ATPase activity appears which catalyzes partial washout of the 18 O. When myosin is saturated with actin, the second activity is responsible for approximately 90% of the ATP hydrolysis while the first is responsible for approximately 10%. The kinetic behavior of the second ATPase in actomyosin makes it appear that the actin and myosin do not dissociate during the hydrolysis and product-release steps. This possibility is currently being tested with the nonfilamentous heavy meroymyosin-factin system.